The conversion of biomass, such as corn, sugarcane or other energy crops, as well as simple sugars, to ethanol is routinely completed through the use of yeast fermentation. However, during yeast metabolism a major byproduct of fermentation is glycerol. Glycerol is formed during anaerobic growth as a way for the yeast to balance its redox state and regenerate NAD+ used as a cofactor during glycolysis. It has been shown that the function of glycerol is likely not as a metabolite itself but rather as an electron sink capturing electrons allowing further growth-linked metabolism to continue. As glycerol is a byproduct with low value, it can be an undesirable by-product of fermentation. It would be beneficial to reduce or eliminate this by-product and further direct more carbon towards desired end-products, such as ethanol.
Several strategies are available in the art for the conversion of glycerol to higher value products though biochemical or other means, but relatively little has been demonstrated for the removal or reduction of glycerol and improvement of overall sugar yield to ethanol or other desired end-products of metabolism. Through engineering of alternate pathways, potentially with the simultaneous reduction or deletion of the glycerol pathway, alternate or replacement electron acceptors for the regeneration of NAD+ can be used during yeast metabolism. Such alternate or replacement electron acceptors could be molecules such as formate or hydrogen.
The elimination of glycerol synthesis genes has been demonstrated but removal of this pathway completely blocked anaerobic growth of the yeast, preventing useful application during an industrial process. Ansell, R., et al., EMBO J. 16:2179-87 (1997); Pahlman, A-K., et al., J. Biol. Chem. 276:3555-63 (2001); Guo, Z P., et al., Metab. Eng. 13:49-59 (2011). Other methods to bypass glycerol formation require the co-utilization of additional carbon sources, such as xylose or acetate, to serve as electron acceptors. Lidén, G., et al., Appl. Env. Microbiol. 62:3894-96 (1996); Medina, V. G., et al., Appl. Env. Microbiol. 76:190-195 (2010). By incorporating a formate pathway as an alternate electron acceptor, glycerol formation can be bypassed and ethanol yield can be increased. The engineering of a pyruvate formate lyase from E. coli, which is capable of converting pyruvate to formate, has been done to increase formate production. Waks, Z., and Silver, P. A., Appl. Env. Microbiol. 75:1867-1875 (2009). Formate engineering in Waks and Silver was done, however, to provide a source of formate in S. cerevisiae for the production of hydrogen by a secondary microorganism, E. coli. Waks and Silver did not combine formate production with the removal of glycerol formation, and the use of formate as an alternate electron acceptor for the reduction of glycerol was not proposed or evaluated. Thus, despite prior efforts to bypass and/or eliminate glycerol production, there exists a need for the engineering of alternate or replacement electron acceptors in a cell to direct more carbon towards desired end-products, such as ethanol.
The importance of engineering alternate or replacement electron acceptors is exemplified in the process of corn mash fermentation. About 16 billion gallons of corn-based ethanol are produced annually, so even small increases in ethanol yield, such as 5-10%, can translate into an extra billion or so gallons of ethanol over current yields. Ethanol production from corn mash typically results in glycerol yields ranging from 10-12 g/L. See Yang, R. D., et al., “Pilot plant studies of ethanol production from whole ground corn, corn flour, and starch,” Fuel Alcohol U.S.A., Feb. 13-16, 1982 (reported glycerol levels to be as high as 7.2% w/w of initial sugar consumed in normal corn mash fermentations or approximately 1.4 g/100 mL using 20% sugar). By reducing or eliminating the glycerol yield in the production of ethanol from corn and re-engineering metabolic processes, increased ethanol yields can be achieved. Additional benefits may be gained in the production of ethanol from corn. Corn mash is a nutrient rich medium, in some cases containing lipid and protein content that can be >3% of the total fermentation volume. As a result of the energy contained in these components, even higher ethanol yields may be achieved than what is predicted using, for example, pure sugar. The additional increases can come from the metabolism of lipids or amino acids in the corn mash medium. The recombinant cells and methods of the invention enable increasing ethanol yields from biomass fermentation by reducing or eliminating glycerol.